Anti cd160 pe

Immunophenotyping was undertaken following APC-conjugated HLA class I tetramer staining of PBMCs at 37°C for 15 min. Details of the tetramers used can be found in Supplementary Table 5. Tetramers were conjugated to APC and a true tetramer response was verified through the lack of background staining by gating all CD3+ T cells, against CD8+ T cells and using a tetramer negative control. Surface staining with the following antibodies was then performed: live/dead blue dye (Invitrogen), anti-CD8 Amcyan (BD Biosciences), anti-CD3 APC-Cy7 (Biolegend), anti-PD-1 PercpCy5.5 (BD Biosciences), anti-CTLA4 PE-Cy7, anti-CD244 FITC, and anti-CD160 PE (Biolegend) before washing and flow cytometric analysis. Memory subset analysis utilized the same panel but included anti-CCR7 FITC (R&D systems) and anti-CD45RA AF700 (Biolegend) and omitted anti-CTLA4, anti-CD244, and anti-CD160. Example flow plots can be found in (Figure S1).

The PBMCs were resuspended in PBS buffer and then incubated with anti-CD4-FITC (Becton Dickinson Biosciences, USA), anti-CD223-APC (R&D Systems, Inc., USA.), anti-PD-1-PE-Cy7 (BioLegend, USA), anti-CD160-PE (BioLegend, USA) and anti-CD244-PerCP-Cy5.5 (BioLegend, USA) antibodies at room temperature for 30 min in the dark. Immunoglobulin IgG isotype-matched antibodies served as the negative controls. The stained cells were analyzed using the FACScan™ system (Becton Dickinson Biosciences, USA).